title: Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors.
abstract: This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2-hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4-green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2-hybrid and glutathione-S-transferase pulldown analyses show Robo4 binding to a Wiskott-Aldrich syndrome protein (WASP), neural Wiskott-Aldrich syndrome protein, and WASP-interacting protein actin-nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4-mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation-promoting factors to promote endothelial cell migration.
authors.affiliation: Molecular Angiogenesis Group, Cancer Research UK, Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Sheldon
authors.forename: Maud
authors.initials: M
authors.lastname: Andre
authors.forename: John A
authors.initials: JA
authors.lastname: Legg
authors.forename: Paul
authors.initials: P
authors.lastname: Heal
authors.forename: John M
authors.initials: JM
authors.lastname: Herbert
authors.forename: Richard
authors.initials: R
authors.lastname: Sainson
authors.forename: Anshula S
authors.initials: AS
authors.lastname: Sharma
authors.forename: Jan K
authors.initials: JK
authors.lastname: Kitajewski
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Heath
authors.forename: Roy
authors.initials: R
authors.lastname: Bicknell
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D011554:Pseudopodia / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000378:metabolism*; D000097905:Roundabout Proteins
chemical_list: D000199:Actins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D051316:Wiskott-Aldrich Syndrome Protein; D000097905:Roundabout Proteins; C462951:ROBO4 protein, human